Discovery of a High Affinity Adenosine A1/A3 Receptor Antagonist with a Novel 7-Amino-pyrazolo[3,4-d]pyridazine Scaffold

Here we describe the design and synthesis of pyrazolo[3,4-d]pyridazines as adenosine receptor (AR) ligands. We demonstrate that the introduction of a 3-phenyl group, together with a 7-benzylamino and 1-methyl group at the pyrazolopyridazine scaffold, generated the antagonist compound 10b, which displayed 21 nM affinity and a residence time of ∼60 min, for the human A1R, 55 nM affinity and a residence time of ∼73 min, for the human A3R and 1.7 μΜ affinity for the human A2BR while not being toxic. Strikingly, the 2-methyl analog of 10b, 15b, had no significant affinity. Docking calculations and molecular dynamics simulations of the ligands inside the orthosteric binding area suggested that the 2-methyl group in 15b hinders the formation of hydrogen bonding interactions with N6.55 which are considered critical for the stabilization inside the orthosteric binding cavity. We, therefore, demonstrate that 10a is a novel scaffold for the development of high affinity AR ligands. From the mutagenesis experiments the biggest effect was observed for the Y2717.46A mutation which caused an ∼10-fold reduction in the binding affinity of 10b.


Supplementary MD simulations and MM-GBSA calculations results
Representative MD simulations results for 10b and 15b against A1R, A3R and A2BR and for 10a, 10c against A1R are included (Table S2).
The MM-GBSA 2-4 method with the OPLS2005 5,6 force field and using a hydrophobic slab 7,8 as implicit membrane model and including waters in the orthosteric binding area, in a radius of 4 Å from the center of mass of the ligand provides a fair ranking for the binding of ligand 10b against the three receptors A1R, A3R or A2BR and for ligands binding 10a-10c inside A1R but did not differentiate 10b and 15b inside A1R (Table S2). This is due the lack of accuracy of the MM-GBSA 3-4 method.

C A2BR -15b
For 15b -A2BR complex a similar binding pose as 10b -A2BR was used. After 100ns molecular dynamic simulation, 15b leaves the binding pocket and enters the membrane area through an opening between TM6 and TM7. The RMSD of this ligand (6.77 ± 0.38 Å) is also indicative of a ligand translation from the starting position. D A3R 15b Figure S2. Docking poses and representative frames, receptor-ligand interaction frequency histograms and RMSD graphs of protein Ca (RMSDprotein;blue plots) and ligand heavy atoms (RMSDligand; red plots) from 100ns-MD simulations of 10b(A)-(B) inside the orthosteric binding area of WT A3R or A2BR and (C),(D) 15b inside the orthosteric binding area of WT A2BR,A3R, respectively. Bars are plotted only for residues with interaction Representative frame from MD simulation S13 frequencies ≥ 0.2. Color scheme: Ligand=cyan sticks, receptor=white cartoon and sticks, hydrogen bonding interactions=yellow (dashes or bars), π-π interactions=green (dashes or bars); hydrophobic interactions=grey; water bridges=blue. For the protein models of A3R, A2BR were used the homology model derived from A2AR (PDB ID 3EML) 4 in complex with a highest-scored docking pose of tested compound.
A similar heterocyclic system with our compounds found in the literature is the 4-(2phenethyl)amino 1-phenylethyl pyrazolo [3,4-b]pyridine which was found selective against A1R. As has already been described in the literature 5 the docking calculations showed that the 1H-pyrazolo [3,4-b]pyridine central scaffold has a π-π interaction with F171, a lipophilic interaction with V87 and L250, and 7-N forms a hydrogen bond with N254. The 4-(2phenethyl)amino substituent is oriented towards the extracellular part of the receptor and interacted with I274 while 1-phenylethyl towards the bottom of the receptor.
The representative ligand N9-methyl,N6-benzyl adenine binds A1R 6 . Our docking calculations show that the adenine ring has a π-π interaction with F171, theimidazole nitrogen forms a hydrogen bond with N254 and thatN6-benzyl is oriented towards the extracellular part of the receptor ( Figure S3). We did not find in the literature any information for the activity of N7-methyl, N6-benzyl adenine against ARs; this compound had been developed against CDK kinases. 7 N9-methyl,N6-benzyl adenine to A1R Figure S3. N9-methyl,N6-benzyl adenine inside the orthosteric binding area of WT A1R; from docking calculations. Color scheme: Ligand=light pink sticks, receptor=white cartoon and sticks, hydrogen bonding interactions=yellow (dashes), π-π interactions=green (dashe).
For the protein models of A1R the experimental structure of the inactive form for A1R in complex with an antagonist (PDB ID 5UEN 8 ) was used for homology modelling. 2.65 ± 0.32 Figure S4. Representative frames from 100ns-MD simulations of (A) 10b inside the orthosteric binding area of WT A1R; (B) 10b inside mutant Y271A A1R. Receptor-ligand interaction frequency histogram and RMSD graphs of protein Ca (RMSDprotein; red plots) and ligand heavy atoms (RMSDligand; blue plots). Bars are plotted only for residues with interaction frequencies ≥ 0.2. Color scheme: Ligand=brown sticks, receptor=white cartoon and sticks, hydrogen bonding interactions=yellow (dashes or bars), π-π interactions=green (dashes or bars); hydrophobic interactions=grey; water bridges=blue. For the protein models of A1R the experimental structure of the inactive form for A1R in complex with an antagonist (PDB ID 5UEN 8 ) was used for homology modelling.
A small TM2 curvature already exists in the model of A3R generated using as template the crystallographic structure of A2AR (PDB ID 3EML) 8 .

Safety
No unexpected or unusually high safety hazards were encountered during the proceeding of this project.

Chemistry
Melting points were determined on a Büchi apparatus and are uncorrected. 1 H NMR spectra and 2D spectra were recorded on a Bruker Avance III 600 or a Bruker Avance DRX 400 instrument, whereas 13 C NMR spectra were recorded on a Bruker Avance III 600 or a Bruker AC 200 spectrometer in deuterated solvents and were referenced to TMS (δ scale). The signals of 1 H and 13 C spectra were unambiguously assigned by using the 2D NMR spectra COSY, NOESY, HMQC, and HMBC. Mass spectra were recorded with a LTQ Orbitrap Discovery instrument, possessing an Ionmax ionization source. The purity of all the target compounds was >95% as ascertained by 1 H-NMR and elemental analysis. Elemental analyses were undertaken using a PerkinElmer PE 240C elemental analyzer and the measured values for C, H, and N were within ±0.4% of the theoretical values. Flash Ethyl 4-bromomethyl-3-isopropyl-1-methyl-1H-pyrazole-5-carboxylate (6a): In a 33% HBr solution in acetic acid (3mL) were added the carboxylate 4a (0.2 g, 1.0 mmol) and paraformaldehyde (98 mg, 3.3 mmol) and the resulting mixture was heated at 90 °C for 3.5 h. The mixture was then poured into ice, made alkaline using NaHCO3 (pH~8) and extracted with ethyl acetate (3x30 mL). The organic solvent was dried (Na2SO4) and evaporated to dryness and the residue was purified by column chromatography (silica gel) using a mixture of dichloromethane/ethyl acetate 10/0.3-10/1 as the eluent to provide 0. Ethyl 4-formyl-3-isopropyl-1-methyl-1H-pyrazole-5-carboxylate (7a): To a solution of the bromide 6a (150 mg, 0.5 mmol) in dry acetonitrile (2 mL) was added under argon Νmethylmorpholine-Ν-oxide (120 mg, 1.0 mmol) and the mixture was stirred for 24 h. The solvent was then vacuum-evaporated, water was added to the residue, and it was extracted with ethyl acetate. The organic phase was dried (Na2SO4) and concentrated to dryness to provide in high purity compound 7a (100 mg, 86 %) as an oil.  (2 mL) was heated at 110 °C for 8h. Upon completion of the reaction (determined by TLC), the excess of phosphorus oxychloride was vacuum-evaporated and the synthesized chloride 9a was used for the next step without further purification due to the relative ease of hydrolysis.
This compound was prepared following a method analogous to that of 9a, starting from 8b. In this case the reaction was completed in2.5 h and the resulting chloride was again not purified.
To a solution of the chloropyrazolopyridazine 9a (42 mg, 0.2 mmol) in absolute ethanol (3 mL), was added benzylamine (66 µL, 0.6 mmol) and the mixture was heated at 80 °C for 2h. The solvent was then vacuum-evaporated, water was added to the residue, and it was extracted with dichloromethane (3x20 mL). The organic extracts were dried (Na2SO4) and evaporated to dryness and the residue was purified by column chromatography (silica gel) using a mixture of cyclohexane/ethyl acetate 4/6 as the eluent, to result in 23 mg (41 %  Ethyl 4-bromomethyl-1-methyl-5-phenyl-1H-pyrazole-3-carboxylate (11b): This compound was prepared following a method analogous to that of 6a, starting from 5b. Yield 48%. Oil.

Cell lines
Stable Flp-In-CHO cell lines expressing the WT A3R were generated and maintained as previously described 13,14 . CHO-K1 cells stably expressing WT A1R, A2AR or A2BR were routinely cultured in Hams F-12, supplemented with 10% Foetal bovine serum (FBS). All were annually checked for mycoplasma infection using an EZ-PCR mycoplasma test kit (Biological Industries, Kibbutz Beit-Haemek, Israel). Production and analysis of the mutant versions of the A1R were as described in ref 15 .

Compounds
NECA was purchased from Sigma-Aldrich and dissolved in dimethyl sulfoxide (DMSO). All compounds used for the in vitro testing were >95% pure by elemental analysis. S22 cAMP accumulation assay cAMP inhibition experiments were performed using a LANCE® cAMP kit as described previously 13,14 . Briefly, Flp-In-CHO cells expressing WT A3R and CHO-K1 cells expressing WT A1R, A2AR or A2BR were seeded in a white 384-well optiplate at a density of 2,000 cells per well and stimulated for 30 min with a range of NECA concentrations, with or without potential antagonists, in the presence of 0.1% BSA and 25 μM rolipram, and 10 μM forskolin (to enable detection of A1R-or A3R-mediated inhibition of cAMP). Since the A2AR and A2BR promote cAMP accumulation, 16 the addition of forskolin was not included when assaying these receptors.

Schild analysis
NECA concentration-dependent response curves were constructed in the presence of either DMSO alone or multiple concentrations of 10b at the A1R and A3R. NECA concentrations ranged from 1 pM to 1 µM. Estimates of the pEC50 values in the presence and absence of the antagonist were determined using the three-parameter logistic equation built into Prism.

NanoBRET assays for binding
NanoBRET competition binding assays were conducted to determine the affinity (pKi) of various potential antagonists at the A1R and A3R as described previously 9 . For both the A1R and A3R, the CellAura fluorescent A3R antagonist (CA200645) with a xanthine amine congener (XAC) structure was used at 20 nM and 5 nM concentration for A1R and A3R, respectively, since it has a slow off rate. Kinetic data was fitted with the 'kinetic of competitive binding' model (see ref. 17 ; built into Prism) to determine affinity (pKi) values and the association rate constant (Kon) and dissociation rates (Koff) for unlabelled A3R antagonists. In agreement with our previous studies 15, 18 we determined the pKd of CA200645 at the A1R to be 18.29 ± 2.4 nM and at the A3R 26.95 ± 3.2 nM. 18 The BRET ratio at 10 min poststimulation was fitted with the "one-site-Ki model" derived from the Cheng and Prusoff correction, built into Prism to determine the affinity (pKi) constant at equilibrium values for all unlabelled antagonists at the A1R and A3Rs.

Data and Statistical analysis
All in vitro assay data were analyzed using Prism 9.0 (GraphPad software, San Diego, CA), with all dose-inhibition curves being fitted using a three-parameter logistic equation to calculate response range and pEC50. Experimental design ensured random distribution of treatments across 96/384-well plates to avoid systematic bias. Agonist stimulation alone was used as an intrinsic control across all experiments. Dose-inhibition/dose-response curves were normalized to forskolinstimulation (A2AR and A2BR) or forskolin inhibition (A1R and A3R) relative to NECA (agonist allowing comparison across AR subtypes), expressed as percentage forskolin inhibition for Gi-coupled A1R and A3R (1 μM or 10 μM, respectively) or stimulation for A2AR and A2BR (100 μM, representing the maximum cAMP accumulation of the system), relative to NECA. For cAMP experiments on A1R mutants, data was normalized to 100 μM forskolin, representing the maximum cAMP accumulation possible for each cell line.

S23
Schild analysis, when using a single concentration of antagonist was performed to obtain, the dissociation constant (pKd) using eq. (1) 19 where D' and D = EC50 values of NECA with and without antagonist present, respectively, [A] = the concentration of antagonist present, and K2 = the affinity constant (KA) of the antagonist used 19 . Receptor binding kinetics was determined as described previously 18 using the Motulsky and Mahan method 17 (built into Prism 9.0) to determine the test compound association rate constant and dissociation rate constant. The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology 20 . Statistical significance (*, p < 0.05;**, p < 0.01;***, p < 0.001, ;****, p < 0.0001) was calculated using a one-way ANOVA with a Dunnett's post-test for multiple comparisons. All statistical analysis was performed using Prism 9.0 on data which were acquired from experiments performed a minimum of five times, conducted in duplicate.

Models of the complexes between A1R or A3R or A2BR with an antagonist
We used for A2AR in inactive state the experimental structure of A2AR in complex with ZM241385 which was resolved by X-ray crystallography (PDB ID 3EML 8 ). We used for A1R in inactive state the experimental structure of A1R in complex with DU172 which as resolved by X-ray crystallography (PDB ID 5UEN 26 ). Their models were retrieved from Adenosinland web service 26 . We superimposed the experimental crystal structure ZM241385 -A2AR complex (PDB ID 3EML) 8 to the WT A3R or WT A2BR models from Adenosiland web-service 27 . (Residue numbers in parenthesis refer to the Ballesteros-Weinstein numbering) 28 . Then, the A2AR (PDB ID 3EML) 8 protein was removed resulting in the WT A3R -ZM241385 or WT A2BR -ZM241385 models used in the study.
In the next step, the WT A1R -PSB36, A3R -ZM241385, A2BR -ZM241385 structures were optimized using the Protein Preparation Wizard implementation in Schrodinger suite 29 . In this process, the bond orders and disulfide bonds were assigned, and missing hydrogen atoms were added. Additionally, N-and C-termini of the protein model were capped by acetyl and N-methyl-amino groups, respectively. All His were treated with imidazoles protonated at Νε except H278 7.43 at Nδ. Each protein was subjected in an all atom minimization using the OPLS 2005 force field 30 with heavy atom RMSD value constrained to 0.30 Å until the r.m.s of conjugate-gradient value reached < 0.05 kcal mol -1 Å -1 .The side chain of V169 5.30 in the WT A3R complex was rotated to fit the conformer as was suggested 31 in order to increase the free space for the accommodation of ligands with bulky substitutions in this area. The WT A1R -DU172, WT A3R -ZM241385, WT A2BR -ZM241385 modelswere utilized for the molecular docking calculations.

Docking calculations
The molecular docking calculations of the 6 tested 7-amino-pyrazolo [3,4-d]pyridazines (Table 1) (the protocol used for the preparation of models of the tested ligands is reported in the Supporting Information) with A1R, A3R, A2BR were performed using GOLD software 32 (GOLD Suite, Version 5.2; Cambridge Crystallographic Data Centre: Cambridge, U.K., 2015. GOLD Suite, version 5.2; Cambridge Crystallogr. Data Cent. Cambridge, U.K., 2015) and ChemScore 33 as the scoring function. The models of WT A1R -DU172, WT A3R -ZM241385, WT A2BR -ZM241385 were used as templates for the molecular docking calculations of the antagonists to the binding area of each of the receptors. Each compound was docked in the binding site of ZM241385 in the A3R-ZM241385 model or DU172 in A1R -DU172, model or ZM241385 in the A2BR-ZM241385 model in an area of 15 Ǻ around the ligand using the experimental coordinates of ZM241385 or DU172 and 20 genetic algorithm runs were applied for each docking calculation. The top-scoring docking poses were used for MD simulations to investigate the binding profile of the 6 tested 7-amino-pyrazolo [3,4d]pyridazines (Table 1) at A1R or A3R or A2BR.

MD Simulations
Each protein-ligand complex was inserted in a pre-equilibrated hydrated 1-palmitoyl-2oleoyl-sn-glycero-3-phosphoethanolamine (POPE) membrane bilayer according to OPM (Orientations of Proteins in Membranes) database 34 . The orthorhombic periodic box boundaries were set 12 Å away from the protein using the System Builder utility of Desmond v4.9 (Schrödinger Release 2021-1: Desmond Molecular Dynamics System, D. E. Shaw Research, New York, NY, 2021. Maestro-Desmond Interoperability Tools, Schrödinger, New York, NY, 2021). The membrane bilayer consisted by ca. 180 lipids and 16,000 TIP3P 35 water molecules. Sodium and chloride ions were added randomly in the water phase to neutralize the system and reach the experimental salt concentration of 0.150 M NaCl. The total number of atoms of the complex was approximately 75,000 and the simulation box dimensions was (88 x 75 x 110Å 3 ). We used the Desmond Viparr tool to assign the amber99sb 36,37 force field parameters for the calculation of the protein, lipids and intermolecular interactions, and the Generalized Amber Force Field (GAFF) 38 parameters for the ligands. Ligand atomic charges were computed using the RESP 39 fitting for the electrostatic potentials calculated with Gaussian03 40 at the HF/6-31G* 41 level of theory and the antechamber of AmberTools18. 42 100 ns MD simulations at constant pressure (NPT) were performed for the 6 ligands (Table 1) in complex with A1R or A3R or A2BR embedded in POPE bilayers using Desmond v4.9 software, the Desmond MD algorithm 43 with amber99sb 44 force field to investigate their binding interactions. The protocol used to calculate interactions and run the MD simulations and the visualization of the trajectories is described in the Supporting Information. Within the 100ns-MD simulation time, the total energy and RMSD of the protein backbone Cα atoms reached a plateau, and the systems were considered equilibrated and suitable for statistical analysis ( Figure S4).

S25
Two MD simulations were performed for each complex using the same starting structure and applying randomized velocities. All the MD simulations with Desmond software were run on GTX 1060 GPUs in lab workstations or the ARIS Supercomputer.

MD simulations protocol
The MD simulation of each protein-ligand complex inside the lipid bilayer was performed using the default protocol provided with Desmond v4.9 program. The MD simulation protocol consists of a series of MD simulations designed to relax the system, while not deviating substantially from the initial coordinates. During the first stage, a simulation was run for 200 ps at 10 K in the NVT ensemble (constant volume, temperature and number of atoms), with solute-heavy atoms restrained by a force constant of 50 kcal mol Å −2 . The temperature was raised to 310 K during a 200 ps MD simulation in the NPT ensemble (constant pressure, temperature and number of atoms), with the same force constant applied to the solute atoms. The temperature of 310 K was used in MD simulations in order to ensure that the membrane state is above the main phase transition temperature of 298 K for POPE bilayers 47 . The heating was then followed by equilibration simulations. First, two 1 ns stages of NPT equilibration were performed. In the first 1 ns stage, the heavy atoms of the system were restrained by applying a force constant of 10 kcal mol -1 Å −2 , and in the second 1 ns stage, the heavy atoms of the protein-ligand complex were restrained by applying a force constant of 2 kcal mol -1 Å −2 to equilibrate water and lipid molecules. In the production phase, the relaxed systems were simulated without restraints in the NPT ensemble for 100 ns. Replicas of the system were saved every 10 ps.
In the MD simulations the Particle Mesh Ewald (PME) 48 method was employed to calculate long-range electrostatic interactions with a grid spacing of 0.8 Å. The SHAKE method was used to constrain heavy atom-hydrogen bonds at ideal lengths and angles 49 . Van der Waals and short-range electrostatic interactions were smoothly truncated at 12 Å 50 . The Nosé-Hoover thermostat 51 was utilized to maintain a constant temperature in all MD simulations, and the Martyna-Tobias-Klein method 52 was used to control the pressure. The equations of motion were integrated using the multistep reversible reference system propagator algorithms (RESPA) 53 integrator with an inner time step of 2 fs for bonded interactions and non-bonded interactions within the cutoff of 12 Å. An outer time step of 6.0 fs was used for non-bonded interactions beyond the cutoff.
The visualization of the MD simulation trajectories was performed using the graphical user interface (GUI) of Maestro and the protein-ligand interaction analysis was carried out with the Simulation Interaction Diagram (SID) tool, available with Desmond v4.9 program. For the calculation of hydrogen bond interactions were considered, a distance 2.5 Å between donor and acceptor heavy atoms, and angle ≥120 o between donor-hydrogen-acceptor atoms and ≥ 90 o between hydrogen-acceptor-bonded atom. Non-specific hydrophobic contacts were identified when the side chain of a hydrophobic residue fell within 3.6 Å from a ligand's aromatic or aliphatic carbon, while π-π interactions were characterized by stacking of two aromatic groups face-to-face or face-to-edge. Water-mediated interactions were characterized when the distance between donor and acceptor atoms was 2.7 Å, as well as an angle ≥ 110 o between donor-hydrogen-acceptor atoms and ≥ 80 o between hydrogen-acceptor-bonded atom.

MM-GBSA calculations
The MD simulation trajectories were used for the calculation of approximate binding free energies of ligand -protein complexes using the 1-trajectory MM-GBSA 3,3 method, the OPLS2005 3,4 force field and 20 waters in the vicinity of the ligand . The MD trajectories were processed with the Python library MDAnalysis 54 13 in order to extract the 20 water molecules closest to any atom in the ligand 55 14 for each of the 501 frames. We applied a dielectric constant εsolute = 1 to the binding area and to account for the lipophilic environment of the protein an heterogeneous dielectric implicit membrane model was used along the bilayer zaxis 39 .
For this, structural ensembles were extracted in intervals of 40 ps from the 20 ns MD simulation for each complex. Prior to the calculations all water molecules, ions, and lipids were removed, except 20 waters in the vicinity of the ligand 55 , and the structures were positioned such that the geometric centre of each complex was located at the coordinate origin.The MD trajectories were processed with the Python library MDAnalysis 53 in order to extract the 20 water molecules closest to any atom in the ligand for each of the 501 frames. During the MM-GBSA calculations, the explicit water molecules were considered as being part of the protein. Binding free energies of compounds in complex with A1R and A3R were estimated using the 1-trajectory MM-GBSA approach. 3,31<sup>15,16</sup><sup>15,16</sup> For the calculation of binding free energy for each complex eqs (1)-(4) were used Δ !"#$ = 〈 complex − protein − ligand 〉 %&'()*+ (1) -= .. − 〈 MM 〉 + Δ solv (2) .. = !&#$*$ + %&/) + 01 (3) The binding free energy for each complex was thus calculated using eq. (5) Δ !"#$ = 〈 coul + Δ 67 〉 − 〈 MM 〉 + ΔΔ solv (5) In eqs (1)-(4) Gi is the free energy of system i, that being the ligand, the protein, or the complex; VMM is the potential energy in vacuum as defined by the molecular mechanics (MM) model, which is composed of the bonded potential energy terms (Vbonded) and nonbonded Coulombic (Vcoul) and Lennard-Jones (VLJ) terms; SMM is the entropy; ΔGsolv is the free energy of solvation for transferring the ligand from water in the binding area calculated using the PBSA model, composed by a polar (ΔGP) and nonpolar (ΔGNP) term; T is the temperature and angle brackets represent an ensemble average. Molecular mechanics energies for Lennard-Jones (VLJ) and Coulombic elecrostatic (Vcoul) were calculated with OPLS2005 3,4 force field ; in these calculations ΔVbonded = 0 as the single trajectory method was adopted and ΔVMM = ΔVLJ and ΔVcoul.The polar part of the solvation free energy was determined by calculations using the Generalized-Born model. 54 17 The nonpolar term was considered proportional to the solvent accessible surface area (SASA), ΔGNP = γ · SASA, where γ = 0.0227 kJ mol −1 Å −2 . The entropy term was neglected since we were interested for binding free energies between ligands in the same series and in this case ΔGbind is termed as effective binding energy, ΔGeff. The post-processing thermal_mmgbsa.py script of the Schrodinger Suite was used which takes snapshots from the MD simulations trajectory and calculates ΔGeff.

Cell viability assays
Human HCT116 colon cancer cell line and PC-3 prostate cancer cell linewere obtained from the American Type Cell Culture (ATCC, Bethesda, Md). Both cell lines were grown in 75cm 2 culture flasks at 37 o C in 5% CO2 using Roswell Park Memorial Institute 1640 medium (RPMI 1640, Gibco) and Dulbecco's modified Eagle's medium F/12 (DMEM/F-12, Gibco) respectively, containing 10% fetal bovine serum (FBS, Gibco). To test the inhibitory activities of compounds using a cell-based assay, HCT116 cells were plated at a density of 1500 per well, while PC-3 cells were plated at a density of 750 per well in a 96-well plate. After 24 h, cells were treated with the indicated compounds in a dose-dependent manner for 72h and 96h (all tested compounds provided clear solutions). MTT [3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide] (Sigma M-5655) was added at a final concentration of 0.5 mg/mL directly to each well for 4 hours at 37 o C. The medium was aspirated and the blue MTT formazan precipitate was dissolved in dimethyl sulfoxide (DMSO). Absorbance was determined in a Powerwave microplate spectrophotometer (Biotek Instruments, Inc.) at 540 nm. Viable cell numbers were determined by tetrazolium conversion to its formazan dye. The IC50 was calculated by Microsoft Excel equation and confirmed by GraphPad Prism (7.0). Each experiment was performed in triplicate and mean values ± SD are reported.